Table of Content

16 February 2019, Volume 52 Issue 4

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Genome-Wide Detection of Selection Signal in Temperate and Tropical Maize Populations with Use of F_ST and XP-EHH

YANG YuXin,ZOU Cheng

Scientia Agricultura Sinica. 2019, 52(4):  579-590.  doi:10.3864/j.issn.0578-1752.2019.04.001

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【Objective】 Maize was first domesticated in tropical areas, but it has been cultivated widely in the temperate regions after natural and artificial selection. Flowering time is not only the key component of the entire growth period, but also a major adaptive trait during the dispersal process from tropical to temperate conditions. Thus, identifying the selected gene regions responsible for the adaptation to temperate zones, and discovering the genes that are involved in flowering time could provide a molecular basis for improving maize and for dissecting its flowering mechanism. 【Method】 We analyzed the haplotype data of 30 temperate and 21 tropical maize inbred lines. High quality SNP (single nucleotide polymorphism) markers were obtained after filtering out SNPs with high missing rates and low allele frequencies. These high quality SNPs were annotated by SNPeff. Principle component analysis (PCA) of the genotypic data of temperate and tropical maize was performed to further validate the population structure of these samples. Using high quality SNP markers that were present in tropical and temperate populations, we calculated the selection signal using the fixation index (F_ST) and cross population extended haplotype homozygosity (XP-EHH) methods. The top 1% of values was used as a significant threshold to identify the candidate selected signals. The candidate selected genes that we selected from temperate and tropical maize were identified based on their SNP annotation. The function of these selected genes was characterized furtherly by the GO enrichment analysis using agriGO. To identify the genes for flowering time that were under selection, bioinformatics databases were examined that contained relevant data on maize. 【Result】 By analyzing the high depth resequencing data, we found 14123408 and 8791673 SNPs in tropical and temperate populations, respectively. The identified SNPs were mainly distributed in the intergenic regions. There were 204752 high quality SNPs that coexisted in temperate and tropical populations. PCA indicated that temperate and tropical maize can be divided into two groups. The top 1% of F_ST value and XP-EHH were 0.3059, 3.2681, and a total of 557 and 1 913 candidate genes were identified by F_ST and XP-EHH methods, respectively. Many candidate genes were highly related to regulation of flowering time, which included ZmCCT9, COL1 and GRMZM2G387528. ZmCCT9 is a vital gene for regulating flowering time, and it negatively regulated the floral activator gene ZCN8, which cause the late flowering time phenotype under long-day conditions. COL1 positively interacts with the FT protein to promote the transition of flowering time to adapt to the long-day environment. Functional annotations of GRMZM2G387528 revealed that it was a phytochrome interacting factor, and interacts with photoperiod gene ZmphyB1. 【Conclusion】 Our study revealed that tropical maize had higher genetic diversity than temperate maize. A series of genes that were under selection during the adaptation to tropical to temperate conditions were predicted, and we further explored the genes that were involved in flowering during this process.

Taste Quality Traits of Sorghum Landraces from Liaoning Province

XIAO MengYing,ZHANG RuiDong,ZHANG Zhuang,XU XiaoXue,CHEN XiaoFei,ZHOU YuFei,KONG FanHua,HUANG RuiDong

Scientia Agricultura Sinica. 2019, 52(4):  591-601.  doi:10.3864/j.issn.0578-1752.2019.04.002

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【Objective】 In this study, 16 sorghum landraces from Liaoning province were used as experimental materials to study the edible quality traits and the relationships among these traits. 【Method】 Nutritional components of 16 sorghum landraces, including amylose, amylopectin, crude fat, crude protein and tannin content in grains, were determined and the edible qualities of these sorghum landraces were evaluated using sensory evaluation. The viscosity of sorghum starch was measured using a Rapid Visco Analyzer (RVA). The differences and the correlations among indices were analyzed. 【Result】 In terms of nutritional quality and sensory evaluation, the overall score of Aizibai 1, Hongkebang, Jinliang 9-2 and Fenzhidahongsui were higher than the other landraces; the mouthfeel, taste, and smell of these two landraces were also better than the others. The content of crude fat in the grains of Aizibai 2 and Zhenbailiang were significantly higher than in other landraces. Contents of crude protein in Hongkebai 1, Hongkebai 2 and Huangkebai landraces were significantly higher than in the others. Starch content in the grains of Dabaigaoliang 1 was significantly higher than in other landraces. Tannin contents in all landraces were less than 0.5%. The taste value of sorghum porridge was negatively correlated with crude protein and tannin content and protein and tannin had negative effects on mouthfeel and taste. Amylopectin had a positive effect on mouthfeel and cold porridge texture. There was a significantly positive correlation between the content of crude fat and the cool paste viscosity, setback viscosity and peak time. The total starch content was positively related to the peak viscosity and the hot pulp viscosity. The crude protein content was significantly positively correlated with the cool paste viscosity, the setback viscosity and the peak time. Amylose content was significantly negatively correlates to the viscosity level and disintegration value of the RVA spectrum. Amylopectin was significantly positively correlated to the viscosity level and disintegration value of the RVA spectrum. RVA was significantly correlated with taste value. The peak viscosity and break down viscosity were positively correlated with the comprehensive score, while the peak time and pasting temperature were negatively correlated with the taste value. 【Conclusion】 There was a close relationship between RVA spectrum characteristic value and taste index. The taste characteristics of sorghum porridge were indirectly reflected by the RVA spectrum characteristic value. Based on comprehensively considering the edible quality, Aizibai 1 and Hongkebang were the ideal sorghum types.

Evaluation of 317 Sugarcane Germplasm Based on Agronomic Traits Rating Data

ZHAO Yong,ZHAO PeiFang,HU Xin,ZHAO Jun,ZAN FengGang,YAO Li,ZHAO LiPing,YANG Kun,QIN Wei,XIA HongMing,LIU JiaYong

Scientia Agricultura Sinica. 2019, 52(4):  602-615.  doi:10.3864/j.issn.0578-1752.2019.04.003

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【Objective】Evaluation of sugarcane germplasm via rapid rating their important agronomic traits could provide reference for their further evaluation hybridization.【Method】Based on the field experiment, 317 sugarcane germplasm resources (2 replicates) were rated and evaluated by selecting five important agronomic traits at maturity stage, which were plant height, stalk diameter, millable stalks, leaf disease degree (comprehensive reflection of natural leaf diseases such as mosaic, rust, brown stripe disease) and general vigor. Each trait was divided into five grades, in which plant height was highest in grade 1, and the shortest in grade 5; similarly, stalk diameter was thickest in grade 1 and finest in grade 5; millable stalk was the most in grade 1 and the least in grade 5; general vigor was the best in grade 1 and the worst in grade 5; leaf diseases were the best in grade 1 and the worst in grade 5. The rating of each trait was accomplished by the members of the rating group. Based on the rating data of agronomic traits, 317 germplasm resources were comprehensively evaluated by statistical methods such as variance, broad-sense heritability, principal component analysis, clustering and discriminant analysis, and the germplasm resources with excellent agronomic traits were screened. Further, the sucrose content of the selected excellent germplasm was evaluated by primary rotation.【Result】The results of variance and broad-sense heritability analysis showed significant differences among 317 sugarcane germplasm in the classification data of agronomic traits (P<0.01) and the broad-sense heritability ranged from 0.61 to 0.72. The stalk diameter performed the highest broad-sense heritability (0.72) and the plant height performed the lowest (0.61). The results of principal component analysis showed that the five agronomic traits could be simplified to one principal component factor, i.e. the comprehensive superposition of five agronomic traits. The information provided by the principal component accounted for 63.57% of the total information, and the characteristic roots were significantly higher than the sum of the characteristic roots of other principal components. Based on the results of principal component analysis, 317 germplasms were classified into five categories, including 20 in the first category, 97 in the second category, 82 in the third category, 80 in the fourth category and 38 in the fifth category. 117 germplasm in the first and the second categories were the best in agronomic traits (36.91% of the total germplasms). The stepwise discriminant analysis of clustering results showed that the average probability of pairing is 95.85%. The clustering results fully reflected the actual performance of germplasm in field experiments. Based on cluster analysis of agronomic traits, the sucrose content of 117 germplasm in Class I and Class II was evaluated. The results showed that the average sucrose content of 117 germplasms ranged from 5.0% to 17.10% from November to next March, the highest was YZ09-1601 (17.1%) and the lowest was CP79-318 (5.0%). There were 43 germplasm with an average sugar content of more than 15.0%, of which 11 were more than 16.0%.【Conclusion】Plant height, stalk diameter, millable stalks, leaf disease and general vigor are important agronomic traits reflecting the field performance of sugarcane germplasm resources. The rapid classification method based on important agronomic traits is a promising alternative for evaluating large number of sugarcane germplasm resources.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Comparison of Hyperspectral Remote Sensing Estimation Models Based on Photosynthetic Characteristics of Winter Wheat Leaves

ZHANG Zhuo,LONG HuiLing,WANG ChongChang,YANG GuiJun

Scientia Agricultura Sinica. 2019, 52(4):  616-628.  doi:10.3864/j.issn.0578-1752.2019.04.004

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【Objective】Photosynthesis is the basis of crop yield and quality formation. Accurate quantitative remote sensing inversion of crop photosynthetic parameters can not only understand the growth and development of crops and the accumulation of organic matter, but also can provide reference for the ecosystem process model based on remote sensing. In order to estimate the photosynthetic characteristic parameters quickly and accurately, the hyperspectral inversion model of three photosynthetic parameters of winter wheat was constructed by combining the original spectrum, three traditional spectral transformation techniques and four simulation methods. The feasibility of hyperspectral inversion of photosynthetic parameters of winter wheat was discussed, and the applicability of different spectra and simulation methods were compared. 【Method】The maximum net photosynthetic rate (Amax), PSⅡ effective photochemical quantum yield (Fv'/Fm') of different leaf ages was obtained under the support of gas exchange and hyperspectral field experiments of winter wheat under different nitrogen application conditions. The photochemical quenching coefficient (qP) and hyperspectral reflectivity were obtained, and the reciprocal, logarithmic and first-order differential transformations of the original hyperspectrum were carried out. According to the results of correlation analysis of three photosynthetic parameters and four spectra, the band whose significant level was better than 0.01 was selected as input variable, and then the partial least square (PLS), support vector machine (SVM), multivariate linear regression (MLR) and artificial neural network (ANN) were used to establish the inversion model of photosynthetic parameters of winter wheat leaves. Based on the determination coefficient (R ²) and root mean square error (RMSE) of modeling and validation process, the simulation accuracy of different models was compared and analyzed.【Result】(1) The correlation analysis of the three photosynthetic parameters and four spectra showed that the sensitive spectral regions of the primitive, reciprocal and logarithmic spectra to the three photosynthetic parameters (Amax, Fv′/Fm′ and qP) were concentrated in the 400-750 nm spectrum range. The sensitive spectral regions of the first derivative spectrum to the three photosynthetic parameters were 470-560, 630-700 and 700-770 nm, respectively. (2) The optimal inversion model of Amax, Fv'/Fm' and qP was composed of MLR model based on reciprocal spectrum, MLR model based on first derivative spectrum and MLR model based on original spectrum, respectively. The R ² of the modeling was 0.75, 0.65 and 0.65, respectively, and the R ² of the validation was 0.73, 0.59 and 0.44, respectively. The results showed that the simulation of Amax and Fv'/Fm' based on hyperspectral method was feasible, the effectiveness of simulated qP needed further be verified. (3) The spectral performance of different transformations was different. Taking PLS simulation Amax as an example, the order of spectral performance was original spectrum > reciprocal spectrum > logarithmic spectrum > first derivative spectrum. (4) The estimation ability of different models was also different. Taking Amax simulation based on original spectrum as an example, the order of estimation ability of different models was MLR > PLS > ANN > SVM.【Conclusion】By comparing four spectra and four simulation methods, the hyperspectral inversion results of three photosynthetic parameters of winter wheat showed that Amax and Fv'/Fm' could be well simulated by hyperspectral method, but hyperspectral interpretation ability to qP was low and further study was needed. The hyperspectral information was sensitive to the photosynthetic parameters of winter wheat and affected by spectral types and simulation methods. It could be used to monitor the dynamic changes of photosynthetic capacity of winter wheat and to provide a basis for understanding the growth of crops.

Effects of Plant Density and Plant Growth Regulator on Stalk Traits of Maize and Their Regulation

XU TianJun,LÜ TianFang,CHEN ChuanYong,LIU YueE,ZHANG YiTian,LIU XiuZhi,ZHAO JiuRan,WANG RongHuan

Scientia Agricultura Sinica. 2019, 52(4):  629-638.  doi:10.3864/j.issn.0578-1752.2019.04.005

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【Objective】Studying and defining the effects of planting density and plant growth regulator on maize stalk traits can provide theoretical basis and technical support for rational close planting, optimizing suitable population structure and realizing high yield of maize. 【Method】 JK968 was used as material. The experiment was conducted to study the effects of planting density and plant growth regulator on maize stalk traits with three planting density treatments, 6.0×10 ⁴ plant/hm ² (D1), 7.5×10 ⁴plant/hm ² (D2) and 9.0×10 ⁴plant/hm ² (D3), and plant growth regulator treatment (ethylene and chloramphenicol compounds, CK). 【Result】 (1) With the increase of planting density, the lodging rate was increased. In the condition of D1 density, the lodging rate of JK968 was 69.1% and 83.4% lower than that of D2 and D3 treatments, respectively. Among them, the lodging rate in D3 was 22.6% and 47.8% higher than D2 and D1, respectively. The ethylene and chloramphenicol (EC) treatment significantly reduced the lodging rate, and the lodging rate D1, D2 and D3 after EC treatment decreased 5.0%, 19.8% and 41.0% than that of the control, respectively. (2) Plant height, ear height, ear position coefficient and center of gravity height were significant different among three plant densities and EC treatment, which showed an increasing trend with the increase of planting density. EC treatment significantly reduced the internodes length below the 6th node of the shoot, increased the internodes length above the 7th node of the shoot. Compared with the control, the height and the ear position coefficient were decreased slightly after EC treatment, while the ear height and gravity center height were significantly decreased. (3) There were significant differences in the bending strength and puncture strength of stem epidermis between different treatments. The stem bending force and rind penetration strength were first increased and then decreased from booting stage to mature stage and reached the maximum value in milk stage. The stem bending force and rind penetration strength of 3rd, 4th and 5th internodes aboveground were significantly different with the increase of planting density and significantly increased after EC treatment. The stem bending force and rind penetration strength of different internodes showed that 3rd > 4th > 5th. (4) The number of grains per panicle and 100-grain weight was significantly decreased with the increase of planting density. After EC treatment, the number of grains per panicle, 100-grain weight and yield increased compared with the control. Under the D1, D2 and D3, the yield of EC treated were 438.8 kg·hm ^-2, 1 041.3 kg·hm ^-2 and 3 376.5 kg·hm ^-2 higher than those of CK, with an increase of 3.6%, 8.2% and 27.8%, respectively. 【Conclusion】 With the increase of planting density, the plant height and height of center of gravity were increased, the basal internodes were belonged, the basal internodes fullness and bending resistance were decreased. EC treatment significantly reduced the internodes length below the 6th node of shoot, significantly increased the internodes length above the 7th node of shoot, slightly reduced plant height, significantly reduced the height of center of gravity and ear height, shortened the length of base internodes and increased the filling degree of base internodes, thus improving the lodging resistance of stem. Therefore, spraying plant growth regulators can significantly increase the bending resistance of maize stalks and the puncture strength of stalk epidermis, significantly reduce ear height, height of center of gravity and lodging rate, which is beneficial to high and stable yield of maize under the conditions of high lodging risk such as frequent wind disasters and high planting density.

PLANT PROTECTION

Cloning and Expression Analysis of the Citrus Bacterial Canker-Related Gene CsPGIP in Citrus

HU AnHua,QI JingJing,ZHANG QingWen,CHEN ShanChun,ZOU XiuPing,XU LanZhen,PENG AiHong,LEI TianGang,YAO LiXiao,LONG Qin,HE YongRui,LI Qiang

Scientia Agricultura Sinica. 2019, 52(4):  639-650.  doi:10.3864/j.issn.0578-1752.2019.04.006

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【Objective】The objective of this study is to clone CsPGIP and analyze its expression characteristics, construct CsPGIP transgenic citrus and evaluate the resistance to citrus bacterial canker (CBC), and to provide a theoretical basis for molecular breeding of citrus bacterial canker.【Method】CsPGIP was annotated from the genomic databases and cloned from Wanjincheng and Calamondin. MEGA6 was used for multiple sequence alignment and phylogenetic tree was constructed. Two online softwares BaCelLo and SignalP 4.0 were used for the prediction of subcellular localization and signal peptide. The predicted result was then demonstrated by GFP transient expression. The expression profile of CsPGIP induced by Xanthomonas citri subsp. citri (Xcc) was also analyzed in Wanjincheng and Calamondin by using qRT-PCR method. The correlation between Xcc infection and CsPGIP expression was analyzed. Genetic transformation of Wanjincheng was conducted by Agrobacterium-mediated method. The over-expressed lines were identified by GUS staining, PCR and qRT-PCR. The phenotypic changes of transgenic and wild-type lines were observed, plant height and leaf phenotype were analyzed. In vitro acupuncture was used to evaluate the resistance of transgenic lines and wild-type lines to citrus bacterial canker. The effect of CsPGIP expression on resistance and susceptibility to citrus bacterial canker was analyzed by statistical analysis of lesion area (LA) and disease index (DI). 【Result】The PGIP cloned from Wanjincheng and Calamondin encodes 328 amino acids, which is 99.39% homology with the reported CsPGIP from Clementina, and contains two typical LRR domains (LRR_1 and LRR_2). In the phylogenetic tree, the genetic distance between CsPGIP and grape PGIP (GSVIVT01033370001) was the closest, and the similarity was 62.97%. It is inferred that CsPGIP and grape PGIP have similar resistance to disease. The prediction of subcellular localization and signal peptide indicated that CsPGIP was a secretory protein, and GFP transient expression proved that CsPGIP located on cell membrane and cell wall, which was consistent with the predicted results. The expression of CsPGIP in canker sensitive plant Wanjincheng and canker resistant plant Calamondin was different after inoculated with Xcc. The expression of CsPGIP was significantly down-regulated in Wanjincheng, but significantly up-regulated and maintained at a high level in Calamondin. It is speculated that CsPGIP was related to resistance to citrus bacterial canker. CsPGIP over-expression vector was constructed and transformed into Wanjincheng, and nine CsPGIP over-expression lines (OE1, OE3, OE4, OE5, OE6, OE9, OE10, OE12 and OE14) were identified as CsPGIP over-expression positive lines by PCR identification and qRT-PCR. Through the phenotypic observation of transgenic lines, it was found that the phenotypes of OE3 and OE14 lines were significantly different from those of wild-type lines. The plant was short, in which OE14 was also abnormal with curly property and greater thickness. The in vitro canker resistance of eight CsPGIP over-expression lines was evaluated. The results showed that the lesion area on the eight CsPGIP over-expression lines was smaller compared to that on the wild-type (24.11%-83.88%), and the lesion area of OE1 was the smallest. In terms of disease index, the disease index of CsPGIP over-expression lines (except OE3) was significantly lower than that of wild-type (23.12%-75.49%), and the decrease of OE1 was the most significant. The above results showed that over-expression of CsPGIP could effectively inhibit the growth of citrus bacterial canker.【Conclusion】CsPGIP is an important gene which can inhibit or reduce the incidence of citrus bacterial canker, and has a great application value in the mechanism study of citrus resistance to bacterial canker. In the same time, it can be used as a candidate gene for molecular breeding of citrus bacterial canker resistance.

Expression and Function Analysis of Endocuticle Structural Glycoprotein Gene LmAbd-2 in Locusta migratoria

JIA Pan,ZHANG Jing,YANG Yang,LIU WeiMin,ZHANG JianZhen,ZHAO XiaoMing

Scientia Agricultura Sinica. 2019, 52(4):  651-660.  doi:10.3864/j.issn.0578-1752.2019.04.007

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【Objective】The objective of this study is to analyze the sequence and expression characteristics of endocuticle structural glycoprotein gene LmAbd-2 that identified from Locusta migratoria transcriptome, explore its function by Hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) based on RNAi with dsLmAbd-2, and to discuss the effect on the formation of endocuticle during L. migratoria molting.【Method】The cDNA sequence of LmAbd-2 was obtained according to the transcriptome database of L. migratoria, and was cloned by reverse-transcription PCR (RT-PCR) and sequenced. The amino acid sequence of LmAbd-2 was translated by ExPASy tool, and its signal peptide and structural domain were analyzed by using SignaIP4.1Server and SMART software, respectively. Meanwhile, the O-linked glycosylation sites were predicted by using NetOGlyc4.0 Server. The homologous sequences of Abd-2 from locust and other insect species were aligned by GENEDOC software and evolutionary tree was constructed by using the MEGA 6.0 software with the neighbor-joining (NJ) method. Expression profiles of LmAbd-2 in different tissues at day 2 of 5th instar nymphs and developmental days of 4th instar nymphs (N4D1-N4D6), 5th instar nymphs (N5D1-N5D8), and the early stage of adults (AD1-AD4) were assayed by using reverse-transcription quantitative PCR (RT-qPCR). The biological function of LmAbd-2 was analyzed by combination of RNAi and H&E staining and TEM methods. 【Result】The results of sequence analysis showed that the length of the LmAbd-2 cDNA is 683 bp and full length of its ORF is 459 bp, encoding 152 amino acids. The functional domain analysis showed that the protein encoded by LmAbd-2 contains one signal peptide and one chitin binding domain (ChtBD4), which belongs to RR-1 subclass of the CPR family. Sequence alignment analysis showed that it is highly conserved among species, and the similarity with SgAbd-2 is 92.5%, and it contains conserved motif (PTPPPIP) in N-terminal and has a potential O-linked glycosylation site (T116) at which glycosylation modification may occur. Phylogenetic tree analysis showed that LmAbd-2 has a close genetic relationship with SgAbd-2 of Schistocerca gregaria. The results of RT-qPCR revealed that LmAbd-2 was highly expressed in the integument, but lower or no expression in other tested tissues, and showed periodic expression during molting. After injection of dsLmAbd-2 on day 3 of 4th instar nymphs and day 4 of 5th instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than those in the control.【Conclusion】An endocuticle structural glycoprotein gene LmAbd-2 was obtained according to the L. migratoria transcriptome database. The protein encoded by LmAbd-2 belongs to RR-1 subclass of CPR family. LmAbd-2 was mainly expressed in integument among all the tested tissues and showed periodic expression at the early stage of 4th instar nymphs, 5th instar nymphs and adults. The results of RNAi suggested that LmAbd-2 was involved in the formation of endocuticle during L. migratoria molting.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Coupling Degree Diagnosis on Suitability Evaluation of Cultivated-Layer Quality for Slope Farmland in Purple Hilly Region of South-Western China

LOU YiBao,SHI DongMei,JIN HuiFang,JIANG GuangYi,DUAN Teng,JIANG Na

Scientia Agricultura Sinica. 2019, 52(4):  661-675.  doi:10.3864/j.issn.0578-1752.2019.04.008

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【Objective】As an important cultivated land resource type in southwest area, the quality of the cultivated-layer of slope farmland in purple hilly is concentrated in the phenomenon of severe erosion, low yield and unstable crop yield. The change of crop yield on the plot scale has a significant hysteresis effect compared with soil quality degradation. Based on the analysis of soil quality of sloping farmland in purple soil with different soil fertility grades, this paper quantitatively analyzed the suitability of crop quality of purple soil slope farmland to crops.【Method】The paper studied the suitability evaluation between crop growth and cultivated-layer for the different land productivity grades by the way of coupling degree model based on statistical analysis and clustering analysis. 【Result】The results showed: (1) the thickness of the cultivated-layer at different land productivity grades for purple slope farmland was from 19 to 21 cm and the effective thickness of the cultivated-layer was the range of 21-43 cm, which indicated that the thickness of cultivated-layer was stable and the thinning phenomenon of effective thickness for cultivated-layer was very serious. The limited factors of crop yield for five levels slope farmland were field slope, thickness of cultivated-layer and effective thickness of cultivated-layer. (2) the characteristics of variation of the three kinds of cultivated-layer types for purple soil slope farmland were very serious. Furthermore, soil in Type I of cultivated-layer indicated such features as weak acidic (pH 6.4) and the highest lower cation exchange capacity (20.99 cmol(+)·L ^-1). Soil in Type II of cultivated-layer was characteristic with smallest field slope (11.3°), the highest thickness of the effective soil layer (38cm) and thickness of cultivated-layer (22 cm), so was the soil available potassium (136.50 mg·kg ^-1). Soil in Type III showed such characteristics as the thinnest effective soil thickness (28 cm), acidic soil (pH 4.8) and the smallest cation exchange capacity (9.19 cmol(+)·L ^-1). The dominant factors affecting the crop yield of purple soil slope farmland included field slope, effective soil thickness, soil acidification and cation exchange capacity. (3) the suitability status of crop and cultivated-layer for the different land productivity grades existed two states, including coordinated development stage and maladjustment decay stage, and four types, which were synchro type, lag type, profit and loss type and common loss type. Crop was more sensitive than quality of cultivated-layer for the coupling coordination degrees under the same land productivity grades of slope farmland, which signified the more yield decrease. Crop-cultivated layer coupling coordination degrees (C_d) for Type I (0.4820） and Type II (0.5207) belonged to the basic coordination development stage and the lag type for crop - cultivated layer, which caused the crop growth was barely suitable. Meanwhile, Crop-cultivated layer coupling coordination degrees (C_d) for Type III (0.3343) was in mild dysregulation stage and profit and loss type, which meant the quality of cultivated-layer for crop growth was moderate unsuitable. 【Conclusion】 The thickness of cultivated-layer was stable and the thinning phenomenon of effective thickness for cultivated-layer was very serious. The suitability status of crop and cultivated-layer for the different land productivity grades existed two states, including coordinated development stage and maladjustment decay stage, and four types, including synchro type, lag type, profit and loss type and common loss type. Slope farmland improvement in purple soil should reduce the slope of the field, increase the thickness of effective soil layer, and adjust the soil pH. These results could provide some useful parameters for suitability diagnosis, regulation and construction of the cultivated-layer of purple soil slope farmland at plots level.

Responses of Crop Yields, Soil Carbon and Nitrogen Stocks to Additional Application of Organic Materials in Different Fertilization Years

GAI XiaPu,LIU HongBin,YANG Bo,WANG HongYuan,ZHAI LiMei,LEI QiuLiang,WU ShuXia,REN TianZhi

Scientia Agricultura Sinica. 2019, 52(4):  676-689.  doi:10.3864/j.issn.0578-1752.2019.04.009

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【Objective】In order to improve crop yield and soil fertility for rotation of winter wheat and summer maize in North China Plain, it is necessary to study the effects of long-term additional application of manure and straw incorporation on crop yield, soil carbon and nitrogen stocks. 【Method】 Taking the key experimental station on ecological environment of Drab Fluvo-aquic soil in Changping, Ministry of Agriculture as the research platform, two application histories (11 years and 27 years) and four application treatments (NPK, chemical fertilizers; NPKM, NPK+22.5 t·hm ^-2manure; NPKM+, NPK+33.75 t·hm ^-2 manure; NPKS, NPK+ straw) were conducted, and yield of winter wheat and summer maize as well as soil carbon and nitrogen stocks were analyzed. 【Result】Results showed that long-term additional application of manure or straw could increase crop yield. Compared with NPK, the yield of wheat and maize could be increased by 18.6%, 15.8%, 3.5% and 39%, 42%, 35% under NPKM, NPKM+ and NPKS, respectively, after 11 years. Meanwhile, yield of wheat and maize could be increased by 41%, 51.5%, 23% and 31%, 33%, 58% under NPKM, NPKM+ and NPKS, respectively, relative to NPK after 27 years. Soil organic carbon (SOC) and total nitrogen (TN) were increased after manure and straw added with the time prolonged. Specifically, stocks of soil carbon and nitrogen were 25-114 Mg·hm ^-2 and 2.2-9.0 Mg·hm ^-2, respectively, after 10 years, which were 29-146 Mg·hm ^-2, 2.5-12.1 Mg·hm ^-2, respectively, after 27 years. Both SOC and TN stocks presented the same trend which increased firstly and then decreased with the increasing of soil depth, reaching a peak at 80 cm. Peaks of SOC and TN pools were 102, 128, 146, 123 Mg·hm ^-2 and 8.3, 9.7, 12.1, 9.1 Mg·hm ^-2 for NPK, NPKM, NPKM+ and NPKS, respectively, after 27 years. However, no significant difference was observed under these treatments after 10 years (P>0.05). Soil ratios of C to N were reduced in different soil layers after two application histories with different organic material application, and soil C/N was obtained stability with the prolonged of fertilization years. Both SOC and TN stocks presented increasing trends with time. Compared with NPK, the accumulation stocks of SOC and TN could increase 5.2%, 11.2%, 9.2% and 21.2%, 26.6%, 38.8% under NPKM, NPKM+ and NPKS, respectively, after 11 years, which could increase 26.3%, 41.1%, 21.8% and 26.2%, 44.9%, 4.0% under NPKM, NPKM+, and NPKS, respectively, after 27 years. For a longer time, additional application of manure was better than straw for promoting SOC accumulation, while it was opposite for TN accumulation.【Conclusion】Based on the conventional fertilization, long-term additional application of manure or straw could increase crop yield, SOC and TN stocks thus promoted soil fertility. A great impact was observed with fertilization years added. In addition, application of manure had a more remarkable effect than straw incorporation, especially for a much long time, but on the contrary for accumulation stocks of TN.

Effects of Application of Nitrogen on Melon Yield, Nitrogen Balance and Soil Nitrogen Accumulation Under Plastic Mulching with Drip Irrigation

XUE Liang,MA ZhongMing,DU ShaoPing,FENG ShouJiang,RAN ShengBin

Scientia Agricultura Sinica. 2019, 52(4):  690-700.  doi:10.3864/j.issn.0578-1752.2019.04.010

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【Objective】The study was aimed at dealing with the problems of fertilization on muskmelon production in field, and the effects of fertilizer on muskmelon yields, soil nitrate accumulation, nitrogen balance of soil nitrogen and nitrogen use efficiency under plastic mulching with drip irrigation were investigated. The purpose of this study was to provide theoretical basis for rational amount of applied nitrogen in muskmelon of fertigation system in Hexi irrigating region.【Method】The field experiment was carried out under plastic mulching with drip irrigation in muskmelon using the split plot design from 2016 to 2017. There was five nitrogen levels, including 0 (N₀), 60 (N₆₀), 120 (N₁₂₀), 180 (N₁₈₀), and 240 kg·hm ^-2 (N₂₄₀), in sub plots to investigate the influence of different fertilizing amount of nitrogen on nutrient absorption and yield of muskmelon. The soil nitrate nitrogen distribution and accumulation were measured at the seeding, vining, fruit-set and mature stages. Furthermore, we also measured the yield of muskmelon at maturity.【Result】The results showed that the yield of the N₁₈₀ treatment was higher than other treatments but it was showed no significant difference compared to N₂₄₀. Nitrogen absorption of melon fruits and nitrogen harvest index were highest under N₁₈₀ treatment. And the higher nitrogen recover efficiency, nitrogen agronomic efficiency and nitrogen physiological efficiency were observed under the treatment of N₁₈₀ for two years, the ranges about 39.59%-40.22%, 56.61-61.44 kg·kg ^-1 and 142.98-152.76 kg·kg ^-1, respectively. And the highest soil NO₃ ^--N content were observed at 0-40 cm soil layers of each treatment at the harvest stage of melon. Meanwhile, the soil NO₃ ^--N contents were reduced with the increasing of depth in 0-100 cm soil profiles, and soil NO₃ ^--N accumulates increased with increasing of the application amount of chemical fertilizer nitrogen. And soil NO₃ ^--N accumulation of each treatment (0-40 cm) accounted for 46.74%-51.84% of total accumulation (0-100 cm). Melon nitrogen uptakes, soil NO₃ ^--N accumulation and nitrogen apparent loss were positively correlated with the nitrogen application rates. Melon nitrogen uptakes, soil NO₃ ^--N accumulation and nitrogen apparent loss accounted for 41.27%-41.36%,42.62%-43.41% and 15.32%-16.02% of the total nitrogen input, respectively.【Conclusion】The best nitrogen application rate was 180 kg·hm ^-2 under plastic cover with drip irrigation in melon production of Hexi corridor oasis irrigation area.

HORTICULTURE

The Characteristics of Sap Flow of Hanfu Apple Trees and Its Response to Environmental Factors

XIA GuiMin,SUN YuanYuan,WANG WeiZhi,WU Qi,CHI DaoCai

Scientia Agricultura Sinica. 2019, 52(4):  701-714.  doi:10.3864/j.issn.0578-1752.2019.04.011

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【Objective】The law of transpiration of fruit trees is embodied in the characteristics of sap flow. The objective of this paper was to study the transpiration and water consumption laws of Malus pumila Mill (‘Hanfu’) in Northeast China, so as to provide a theoretical basis for the development of suitable irrigation system.【Method】In this experiment, the sap flow rates from young fruit stage to the deciduous stage of Hanfu apple trees were continuously monitored by the thermal dissipation probes (TDP) from May to October in 2017, and the meteorological data were continuously recorded by automatic weather station in this orchard. The characteristics of sap flow in Hanfu apple trees and their relationship with environmental factors were analyzed, and then a regression model between sap flow rate and environmental factors was established.【Result】The results showed that the rates of sap flow in Hanfu apple trees were higher in the daytime than that at night. The sap flow rates of Hanfu apple trees at night were stable, the change of sap flow rate was gentle and closed to zero between 0 o’clock and the sunrise, and the high sap flow rate level was still maintained in the period from the sunset to 0 o’clock of the next day. During the growth periods of fruit trees, the start-up and fall time of sap flow was concentrated, and the time of reaching the peak value was dispersive. The proportion of nocturnal sap flow was October > September > May > June > August > July. The proportion of nocturnal sap flow in October was 33.69%, but only 4.57% in July. The correlation degree between environmental factors and sap flow of Hanfu fruit trees was as follows: solar radiation > atmospheric temperature > wind speed > water vapor pressure difference > relative humidity > 30cm soil layer temperature. The multivariate regression equation of sap flow rate and environmental factors of Hanfu fruit tree was: V=6.441+0.012Rn+1.874T–0.577Ts,_5cm+1.915Ws–9.766VPD–0.362RH, and the correlation coefficient of R ² was 0.842. The correlation coefficient between daily sap flow and 10cm soil moisture content was 0.521, which was not significantly correlated with the other layers soil moisture content. 【Conclusion】 The results showed that in the cold area of Northeast China, the transpiration of Hanfu apple trees was large from June to September, and the transpiration was affected by solar radiation, wind speed and other environmental factors. It should be noticed that in the fruit expansion period, especially in July and August, the irrigation should be carried out timely. Irrigation time should avoid the strongest solar radiation time, and be arranged before sunrise or after sunset to reduce water loss caused by evaporation.

Effects of Different Soil Textures on the Degree of Replanted Disease of Malus hupehensis Rehd.

SHENG YueFan,WANG HaiYan,QIAO HongYuan,WANG Mei,CHEN XueSen,SHEN Xiang,YIN ChengMiao,MAO ZhiQuan

Scientia Agricultura Sinica. 2019, 52(4):  715-724.  doi:10.3864/j.issn.0578-1752.2019.04.012

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【Objective】The occurrence degree and mechanism of apple replant disease (ARD) in the apple orchards with different soil textures were studied with the Malus hupehensis Rehd. seedlings as experimental material, so as to adopt appropriate control measures according to the severity of ARD.【Method】Pot experiment including six treatments, which were the replanted sandy loam soil and its methyl bromide fumigation treatment, the replanted loam soil and its methyl bromide fumigation treatment, and the replanted clay loam soil and its methyl bromide fumigation treatment, was carried out. And the soils were from Laizhou, Yantai. The biomass, root protective enzymes activities and malonaldehyde (MDA) contents of M. hupehensis seedlings, changes of the number of soil bacteria, fungi and actinomycetes, and soil enzyme activities were measured by conventional method. The number of the F. oxysporum was detected by a real-time quantitative PCR detection system. Because of the three different soil conditions, the differences of the above indicators between the three soil treatments of methyl bromide fumigation and their replanted soil treatments were used to indicate the degree of ARD. The greater of the difference, the more serious the ARD was. At the same time, the content of phenolic acids of three kind of soils was determined by high performance liquid chromatography.【Result】 Compared with their respective controls, the dry weight of plants in the methyl bromide fumigated clay loam, sandy loam soil and loam soil increased by 98.9%, 87.9%, and 54.4%, respectively. From the results, we could see that the most difference occurred between the replanted soil and its methyl bromide fumigation treatment in clay loam soil, which indicated that the degree of ARD in clay loam soil was the most serious. Compared with their respective controls, the plants treated with methyl bromide fumigation in clay loam, sandy loam and loam significantly increased the root activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and decreased the MDA content. The activity of SOD was 2.63, 1.80 and 1.53 times higher than that of the control, POD activity was 3.02, 2.01 and 1.62 times higher than that of the control and CAT activity was 3.25, 2.61 and 2.11 times higher than that of the control, respectively. The difference of root protective enzymes was the greatest in clay loam, which indicated that root stress was more serious in the replanted clay loam soil. Compared with their respective controls, the root respiration rates of clay loam, sandy loam and loam treated with methyl bromide fumigation increased by 91.3%, 69.4% and 36.0%, respectively. MDA content decreased by 51.3%, 48.9% and 33.1%, respectively. Urease activity decreased by 68.2%, 64.2% and 54.4%, respectively, and phosphatase activity decreased by 25.6%, 18.6% and 8.18%, respectively, compared with their respective controls. The amount of fungi treated with methyl bromide fumigation in clay loam, sandy loam soil and loam soil decreased by 85.8%, 58.1% and 72%, respectively. The copy number of F. oxysporum decreased by 87.5%, 70.1% and 66.3%, respectively, and the copy number of F. oxysporum was the highest in the replanted clay loam soil. Under replanted conditions, the total phenolic acid content in clay loam soil was the highest, which was 41.30 mg·kg ^-1.【Conclusion】 Among three soils of different texture, the degree of ARD was the most serious in clay loam soil on account of the highest total content of phenolic acid, the most harmful fungi mainly in F. oxysporum and the most serious root stress, while the degree of ARD in loam soil was the slightest.

FOOD SCIENCE AND ENGINEERING

Effects of Ultrasound on the Sugar Permeability Effect, Drying Energy Consumption and Quality of Kiwifruit Slices

ZENG XiangYuan,ZHAO WuQi,LU Dan,WU Ni,MENG YongHong,GAO GuiTian,LEI YuShan

Scientia Agricultura Sinica. 2019, 52(4):  725-737.  doi:10.3864/j.issn.0578-1752.2019.04.013

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【Objective】 This research aimed to investigate the effects of ultrasonic on the sugar permeability effect, drying energy consumption and quality of kiwifruit slices and its mathematical model and then the process parameters was optimized to provide a theoretical basis for the application of ultrasonic osmosis technology to produce high quality and low energy kiwifruit slices.【Method】 Taking the kiwifruit as the raw material, the ultrasonic density, time, temperature and sucrose concentration were selected as the factors, and the solids gain (SG), water loss (WL), unit energy consumption, titratable acid, sugar content, color difference (ΔE), L*, a*, b*, hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness, resilience, chlorophyll preservation rate, vitamin C preservation rate and soluble solid were selected as indicators. The box Benhnken test with four factors was designed, and the characteristic index for evaluating the quality of the kiwifruit slices was screened out using factor analysis. The two polynomial regression equation model of the unit energy consumption and the quality characteristic index of the kiwifruit slices were established. The primary and secondary factors and interactions of the effects were analyzed, and the optimal parameters of the ultrasonic infiltration process of kiwifruit slices were optimized and verified. 【Result】 The characteristic index for evaluating the quality of the kiwifruit slices were resilience, ΔE, sugar content, WL, titratable acid and vitamin C preservation rate. The regression model of the energy consumption and quality characteristics of kiwifruit slices was statistically significant (P<0.05). The order of the effect of each factor on sugar content was sucrose concentration>time>temperature>ultrasonic density, and the interactions between time and temperature, temperature and sucrose concentration, sucrose concentration and ultrasonic density were extremely significant. The interaction between temperature and ultrasonic density was significant. The order of the effect of each factor on WL was time>ultrasonic density>sucrose concentration>temperature, and the interactions between time and temperature and time and ultrasonic density were significant. The order of the effect of each factor on unit energy consumption was sucrose concentration>time>ultrasonic density>temperature, and the interaction between temperature and sucrose concentration was significant. The order of the effect of each factor on resilience was time>sucrose concentration>temperature>ultrasonic density, and the interaction between sucrose concentration and ultrasonic density was significant. The order of the effect of each factor on ΔE was sucrose concentration> temperature=ultrasonic density>time. The order of the effect of each factor on titratable acid was time>ultrasonic density>sucrose concentration>temperature, and the interaction between time and ultrasonic density was significant. The order of the effect of each factor on the retention rate of vitamin C was sucrose concentration> temperature>time>ultrasonic density. The optimal process parameters for ultrasonic infiltration of kiwifruit slices were ultrasonic time of 58 min, temperature of 47℃, sucrose concentration of 40 °Brix, and ultrasonic density of 0.7 W·mL ^-1. Under these conditions, the unit energy consumption of kiwifruit slices was 18.15 kJ·g ^-1, the recovery was 0.172, ΔE was 15.51, the sugar content was 35.03%, the WL was 27.85%, the titratable acid was 1.58%, and the vitamin C retention rate was 92.23%.【Conclusion】 The factor analysis method could extract the characteristic indexes for evaluating the quality of kiwifruit slices. The established quadratic polynomial regression model could be applied to analyze and to predict the effects of ultrasonic treatment parameters on the osmotic effect, drying energy and quality of kiwifruit slices. Ultrasonic treatment had the advantages of fast sugar infiltration rate, little damage, lower energy consumption and better texture quality. Ultrasonic treatment could be used in the kiwifruit infiltration process.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

The Screening and Identification of LncRNA Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5

JIN MEI,ZHANG LIJUAN,CAO QIAN,GUO XinYing

Scientia Agricultura Sinica. 2019, 52(4):  738-754.  doi:10.3864/j.issn.0578-1752.2019.04.014

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【Objective】 The aim of this study was to screen out the LncRNA associated with villus growth in Liaoning cashmere goat skin fibroblasts, and provide basic data for the study of the function and mechanism of LncRNA related to villus growth. 【Method】 The total RNA of MT and FGF5 treated Liaoning cashmere goat skin fibroblasts was extracted, and the total RNA extracted was detected by total RNA electrophoresis detection, sequencing data quality evaluation, mapping comparison and inter-sample correlation test. The differentially expressed LncRNA was screened and its target gene was predicted. The LncRNA related to villus growth was screened by GO and KEGG enrichment analysis, and the target LncRNA was verified by Real-time PCR. 【Result】 (1) The total RNA quality of the sample showed that the RNA was in good integrity, the GC content was relatively high, the sequence was stable, and the expression level between samples was high, which met the sequencing requirements.(2) Screening of differentially expressed LncRNA showed that there were 32 differentially expressed LncRNA in 1.0g·L ^-1 24h group, 4 of which were up-regulated and 28 of which were down-regulated. There were 10 differentially expressed LncRNA in 0.2g·L ^-1 24h group, 4 of which were up-regulated and 6 were down-regulated. There were 113 differentially expressed LncRNA in the 0.2g·L ^-1 72h group, of which 5 were up-regulated and 108 were down-regulated. There were 164 differentially expressed LncRNA in the 10 ^-4g·L ^-1 24 h group, of which 70 were up-regulated and 94 were down-regulated. There were 189 differentially expressed LncRNA in the10 ^-4g·L ^-1 72 h group, of which 78 were up-regulated and 111 were down-regulated. There were 123 LncRNA differentially expressed in the 10 ^-6g·L ^-1 24 h group, among which 27 up and 96 down.(3) Target gene GO enrichment analysis showed that the 1.0g·L ^-1 24h group differentially expressed LncRNA target gene enrichment in GO's negative regulation of transcription from RNA polymerase II promoter; 0.2g·L ^-1 24h group did not differentially express LncRNA target gene enriched GO term;0.2g·L ^-1 72h group Differentially expressed LncRNA target gene enrichment in GO's cellular metabolic process biological_process, binding molecular_function, FGF5 treatment group only10 ^-4g·L ^-1 72 h group differentially expressed LncRNA target gene enriched in cell cellular_component, cell part cellular_component, intracellular cellular_component, binding molecular_function and other six items. (4) Target gene KEGG enrichment analysis showed that the differential expression of LncRNA target gene in 1.0g·L ^-1 24h group was enriched in Steroid biosynthesis pathway; in 0.2g·L ^-1 24h group, there was no differential expression of LncRNA target gene enrichment Pathway term; 0.2g·L ^-1 72h group differentially expressed LncRNA target gene enrichment in Cell cycle, DNA replication, Steroid biosynthesis, TNF, Nod-like receptor, NF-kappa B and other signaling pathways, in which TNF and NF-kappa B signaling pathways are involved in villus growth. In FGF5-treated group, differentially expressed LncRNA targets in 10 ^-4g·L ^-1 72 h group The gene was significantly enriched into nine path termes such as Fanconi anemia pathway, Huntington's disease, Metabolic pathway, Aminoacyl-tRNA biosynthesis, among which Metabolic pathway was associated with villus growth; the differentially expressed LncRNA target gene in 10 ^-4g·L ^-1 24 h group had no significant enriched pathway term;10 ^-6g·L ^-1 24 h The differentially expressed LncRNA target genes were only enriched in the Taste transduction pathway. (5) There are two LncRNA corresponding to the target genes TNFα, TNFAIP3 (A20), NFKBIA (IkBα), NFKB2 and IL8 enriched in NF-κB and TNF signaling pathways, respectively (Gene ID): XLOC_005914; XLOC_018763; There are four LncRNA corresponding to the target genes in the Metabolic pathway, namely (Gene ID): XLOC_011424, XLOC_009522, XLOC_009063, XLOC_01115. Real-time PCR results showed that LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 were consistent with high-throughput sequencing results. 【Conclusion】 LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 may increase the density and length of cashmere by regulating NF-κB, TNF or Metabolic signaling pathways related to villus growth, and thus improve the yield and quality of cashmere in Liaoning cashmere goat.

Tissue Expression and Polymorphism of Sheep SMAD1 Gene and Their Association with Litter Size

TIAN ZhiLong,TANG JiShun,SUN Qing,WANG YuQin,ZHANG XiaoSheng,ZHANG JinLong,CHU MingXing

Scientia Agricultura Sinica. 2019, 52(4):  755-766.  doi:10.3864/j.issn.0578-1752.2019.04.015

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【Objective】Sheep litter size is a very complex trait influenced by many factors. Ovulation is one of the most important factors. SMAD protein plays an important role in the mammalian follicular development as well as growth and differentiation process of granulosa cell. To reveal the relationship between the SMAD family member 1 (SMAD1) gene and the litter size of sheep and its mechanism affecting litter size, meanwhile to provide a scientific basis for sheep molecular breeding, we researched the expression pattern of SMAD1 in Small Tail Han sheep with different fertility and analyzed the relationship between polymorphism and the litter size in sheep, based on the previous sheep genome resequencing data. 【Method】First, reverse transcription PCR was used to detect the expression of SMAD1 gene in Small Tail Han sheep (brain, cerebellum, hypothalamus, pituitary, uterus, ovary, oviduct, heart, liver, spleen, lung, kidney, adrenal glands, thyroid, large intestine, small intestine, pancreas, rumen, and kidney fat) tissues with different lambing abilities. Then, Real-time PCR was performed to investigate the expression of SMAD1 gene in Small Tail Han sheep reproduction tissues (hypothalamus, pituitary, uterus, ovary, and oviduct) with different lambing abilities. Multiparous (Small Tail Han sheep (n=380), Hu sheep (n=101), Cele black sheep (n=52)) and uniparous (Sunite (n=21), Tan sheep (n=22), Prairie Tibetan sheep (n=161)) sheep breeds were selected and Sequenom MassARRAY ^?SNP assay was applied to genotype five single nucleotide polymorphism sites (SNPs) of SMAD1 gene. Then the association was analyzed between SMAD1 and litter size in Small Tail Han sheep by using SPSS 19.0. 【Result】The results of RT-PCR revealed that SMAD1 gene expressed in 19 tissues. The qPCR showed that the expression of SMAD1 was higher in ovary (P<0.01), hypothalamus (P<0.05) and pituitary (P<0.05) of uniparous Small Tail Han sheep than that in multiparous sheep. From genotyping, this study found that the genotype frequencies and allele frequencies of the g.12485895A>G, g.12487558G>A and g.12487190G>T of SMAD1 gene were significantly different between uniparous and multiparous sheep (P<0.05). However, g.12508874T>C and g.12487467A>G had no significantly different between uniparous and multiparous sheep (P>0.05). Population genetic analysis indicated that g.12485895A>G, g.12487558G>A, g.12508874T>C, g.12487467A>G, and g.12487190G>T loci were at moderate polymorphism (0.25<PIC<0.5) in Small Tail Han sheep, Hu sheep, Prairie Tibetan sheep, Cele black sheep, Sunite and Tan sheep, while g.12508874T>C was at low polymorphism (PIC<0.25) in Tan sheep. The χ ² test indicated that the g.12485895A>G and g.12487467 A>G were not under Hardy-Weinberg equilibrium (P<0.05) in Small Tail Han sheep and Prairie Tibetan sheep. g.12487558 G>A was not under Hardy-Weinberg equilibrium (P<0.05) in Small Tail Han sheep and Hu sheep. g.12487190 G>T was not under Hardy-Weinberg equilibrium (P<0.05) in Prairie Tibetan sheep. Other loci were under Hardy-Weinberg equilibrium (P>0.05) in six sheep breeds. Association analysis indicated that the polymorphism of g.12487190 G>T had significant correlation with the litter size in Small Tail Han sheep, while the litter size of TT was higher than GT and GG in the first three births (P<0.05). However, the polymorphism of g.12485895A>G, g.12487558G>A, g.12508874T>C and g.12487467A>G had no significant correlation with the litter size in Small Tail Han sheep. The combination of SMAD1 gene g.12487190G>T and FecB (A746G) gene showed that the number of lambs in AA-GG, AA-GT, AA-TT genotypes was significantly low than that of other genotypes (P<0.05), respectively. 【Conclusion】Therefore, it could be concluded that SMAD1 might be the key gene for litter size and the g.12487190G>T locus might provide a basis for litter size trait selection in sheep.

Recombinant Expression and Antimicrobial Activity of Apidaecin in Apis cerana cerana

CHEN WenFeng,WANG HongFang,LIU ZhenGuo,ZHANG WeiXing,CHI XuePeng,XU BaoHua

Scientia Agricultura Sinica. 2019, 52(4):  767-776.  doi:10.3864/j.issn.0578-1752.2019.04.016

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【Objective】In order to study the recombinant expression of antimicrobial peptide Apidaecin of Apis cerana cerana in Bacillus subtilis, verify whether the purified recombinant antimicrobial peptide Apidaecin has antibacterial activity in vivo and in vitro or not, and to provide a theoretical basis for the development of new antimicrobial peptide preparations with safe antibacterial and immunomodulatory functions.【Method】The experiment used Apidaecin in Apis mellifera ligustica as templates to clone the antimicrobial peptide Apidaecin in A. c. cerana, and his-pHT43/Apidaecin expression vector was successfully constructed. In addition, the competent cells were successfully prepared according to the method provided by MoBiTec. The cells were prepared and used on demand to ensure the activity. Protein was isolated and purified using 6×His-Tagged Protein Purification Kit (soluble protein), and the Easy II Protein Quantitative Kit (BCA) was used for concentration determination. The purified recombinant antimicrobial peptide Apidaecin acted on Escherichia coli K88. The disk diffusion test and minimum inhibitory concentration (MIC) were conducted in vitro. In vivo, mice were used as experimental animal models, mice in the experimental group were injected with recombinant antibacterial peptide Apidaecin by intraperitoneal injection, mice in the negative control group were injected with normal saline of the same volume, and the positive control group was injected with the same volume of cefotaxime, then artificially infected with E. coli K88. After 24 hours of infection, the mice were dissected, intestinal samples and blood samples were obtained, and the protective effect of Apidaecin on the mice infected with E. coli K88 was discussed from the perspective of intestinal barrier and immune function. Serum immunoglobulin (IgA, IgG, IgM) and intestinal sIgA levels were determined by using ELISA Kit in the experiment. In addition, the expression levels of intestinal tightly-connected protein genes (claudin-1, ZO-2) and intestinal cytokines (proinflammatory cytokines TNF-α, IFN-γ, IL6 and anti-inflammatory factor IL10) were determined by qRT-PCR. 【Result】The Apidaecin in A. c. cerana was cloned, containing 183 bp, encoding 60 amino acids, including signal peptide sequence of Apidaecin, a basic RR dipeptide and amino acid sequence of Apidaecin (containing 8 highly conserved amino acid sequences). The protein encoded is named AccApidaecin, its molecular weight is 15.6 kD and isoelectric point is 5.33. About 20 mg antimicrobial peptides could be purified from 1 L supernatant at the concentration of IPTG was 3 mmol·L ^-1, induction temperature was 30℃, and inducing time was 12 h. The recombinant antibacterial peptide Apidaecin showed obvious antibacterial ring in vitro compared with the negative control, and the measured MIC was 10 mg·L ^-1. In mice, there were significant differences in immunoglobulin between the bacterial test group injected with recombinant antimicrobial peptide Apidaecin by intraperitoneal injection and the bacterial test group injected with saline (P<0.05), which indicated that recombinant Apidaecin could effectively alleviate the increase of immunoglobulin content in mice caused by E.coli K88. The gene expression level of intestinal related proteins in mice was also significantly different (P<0.05), which indicated that the recombinant Apidaecin could effectively protect the intestinal tract of mice infected with E. coli K88.【Conclusion】The antimicrobial peptide Apidaecin in A. c. cerana was successfully expressed in B. subtilis. The purified antibacterial peptide Apidaecin in A. c. cerana has a good antibacterial effect on E. coli K88 in vitro. In addition, it can be injected into mice through abdominal cavity to improve the immune function of mice and effectively resist the infection of E. coli K88 to mice.